Inhibition of steroid-protein interactions by dicyclohexane derivatives.

Sixteen dicyclohexane derivatives including the parent compound d,1-3,4-bis (4-oxocyclohexyl)-hexane (PRDX) have been synthesized and studied for putative interference with androgen binding to transport proteins, metabolizing enzymes, and receptors from rat tissues. Several of these analogues inhibited competitively the binding of dihydrotestosterone to ABP, the epididymal androgen transport protein. One compound had an affinity for ABP as high as Kd = 70 nM. Some dicyclohexanes also inhibited the aromatase enzyme which catalyses conversion of androgens into estrogens, as well as the NADPH-dependent, particulate form of 3 alpha(beta)-hydroxysteroid dehydrogenase, the enzyme that converts dihydrotestosterone into 5 alpha-androstanediol. For both enzymes the inhibition potency Ki of PRDX was about equal to the Km of the substrate. All of these interactions were specific in that they were modulated by single substitutions on the dicyclohexane molecule and they did not occur with other steroid binding proteins such as 5 alpha-reductase and the intracellular androgen receptor. A conformational study showed that dicyclohexanes can assume a 'steroidoid' conformation that differs from the crystal structure and which could account for the specific interactions with the steroid binding sites described here.

Effects of dicyclohexane derivatives on androgen metabolism in the rat prostate.

Dicyclohexane derivatives, which inhibit the binding of testosterone and dihydrotestosterone (DHT) to the androgen-binding protein (ABP) of rat epididymis without interfering with their binding to the androgen receptor, show a similar selectivity in their effects on androgen metabolism. Their ability to inhibit the aromatization of testosterone has been reported previously. This paper demonstrates that they are potent inhibitors of 3 alpha(beta)-hydroxysteroid:NAD(P)+ oxidoreductase activity (3-HSD) in the particulate fraction from rat prostate gland; the values of Ki for their inhibition of this enzyme are similar to that of the Km for DHT as substrate. The dicyclohexane derivatives are markedly less effective against the cytosolic NADPH-dependent 3-HSD, and they do not appear to inhibit testosterone 5 alpha-reductase activity. These characteristics are likely to complicate the proposed use of the dicyclohexane derivatives as probes for the role of ABP in vivo. However, they may be of interest in the study of structure-activity relationships in androgen-metabolizing enzymes, particularly in the examination of the different forms of 3-HSD.

Progestin binding in benign hyperplastic prostatic tissue.

The identification of saturable binding sites for promegestone (17 alpha, 21-dimethyl-19-norpregna-4,9-diene-3,20-dione) in human prostatic cytosol as progesterone receptors is complicated by some differences in binding behaviour between progesterone and promegestone. These differences seem to result from the presence in cytosol of a second class of progestin-binding sites, which has a higher affinity for progesterone than for promegestone.

Saturable binding of [3H]-promegestone in sheep liver cytosol is not to receptors for progesterone or glucocorticoids.

Saturable, high affinity binding of [3H]-promegestone has been observed in sheep liver cytosol. In two tissues specimens the Kd values were 5.9 and 6.3 nmol/l, and the concentrations of binding sites were 84.5 and 66.2 pmol/g wet tissue respectively. The synthetic steroids methyltrienolone and ORG 2058 were capable of some degree of competition. Progesterone was a weak competitor; cortisol, dexamethasone and testosterone were ineffective. Bound promegestone in liver cytosol was taken up by liver nuclei, but not by sheep uterine nuclei. These promegestone-binding sites are neither progesterone or glucocorticoid receptors.

Effect of sodium molybdate on the interaction of androgens and progestins with binding proteins in human hyperplastic prostatic tissue.

A reliable measurement of steroid hormone receptors is essential for attempts to correlate receptor levels with response to endocrine therapy in prostatic carcinoma. Evidence that receptors in many tissues are stabilized by sodium molybdate prompted the examination of the effects of this salt on the measurement of steroid-binding sites in human prostatic tissue. The presence of molybdate (10 mmol/l) during tissue homogenization, cytosol or nuclear extract preparation and binding-site assay led to a threefold increase in the amount of high-affinity androgen binding detected in cytosol, and a slight increase in the number of cytosol progestin-binding sites. The apparent binding affinity for steroids was increased in both cases. No effect of molybdate was observed on androgen-binding sites in nuclear extracts.

Androgen binding in cytosols and nuclei of human benign hyperplastic prostatic tissue.

Androgen binding sites have been detected in cytosols and nuclear extracts from human benign hyperplastic prostatic (BPH) tissue with exchange assays using [3H]methyltrienolone and [3H]5alpha-dihydrotestosterone respectively. The concentrations of binding sites and the equilibrium dissociation constants for the [3H]steroid-receptor interactions have been determined and the specificity of the binding has been examined. The observed properties of the binding sites are consistent with those characteristic of androgen receptors. The binding sites are present in nuclear extracts from all BPH tissue samples examined to date. The amount of binding detected in the nuclear fraction is higher than that found in the cytosol.